Reduced glutathione (GSH) plays a critical role in a multitude of biochemical processes, and disturbances in its homeostasis are implicated in the etiology and progression of a number of diseases. The initial step in the turnover of this tri-peptide in all mammalian cells is its transport into the extracellular space; however, the transport systems that mediate GSH efflux remain poorly defined. In the liver, a major site of GSH synthesis, GSH is released at high rates into both blood plasma and bile. GSH transport into bile functions as a driving force for bile secretion and plays an important role in hepatic detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin. GSH is also released across the sinusoidal membrane into blood plasma for delivery to other tissues. Our recent studies provide important insight into molecular mechanisms of GSH transport. In particular, we demonstrated that oatp1, the sinusoidal organic solute transporter, functions as a GSH/organic solute exchanger. This finding not only identifies the energy coupling mechanism for oatp1, but also elucidates a pathway for GSH release into blood plasma, as well as a novel function for GSH. Moreover, we demonstrated that Ycf1p, the yeast orthologue of mammalian mrp proteins, mediates ATP-dependent, low-affinity GSH transport, indicating that mrp may mediate GSH efflux in mammalian cells. The overall goals of the proposed studies are to identify and characterize GSH and glutathione S-conjugate transport mechanisms, and in particular to test the hypothesis that the oatp- and mrp-family of transporters mediate cellular GSH release. Our specific aims are: (1) Test whether oatp2, a recently cloned transporter that is homologous to oatp1, also mediates GSH/organic solute exchange. (2) Establish the kinetics and specificity of GSH transport on oatp1, and on oatp2 if this transporter is also found to function as a GSH exchanger. (3) Test whether GSH is a substrate or a co-substrate for mrp2, and if so; a) define the energetics of transport on this canalicular membrane protein, b) compare these parameters with those for GSH transport on yeast Ycf1p, and c) examine whether GSH is also a substrate for mrp3 and mrp6, putative hepatocellular lateral membrane proteins; and (4) Continue to define the role of the intrahepatic &#61543;-glutamyl cycle in the disposition of glutathione S-conjugates and their conversion to the corresponding mercapturic acids by examining mechanisms of mercapturic acid transport.